Recently there has been an increased interest in predictive, preventative, and particularly personalized medicine which requires diagnostic tests with high reproducibility, sensitivity and specificity. Lateral Flow (LF) assay devices incorporate such diagnostic test and are a well-established technology in Point-of-Care (POC) diagnostics. When compared with other diagnostic techniques such as ELISA and PCR/Q-PCR, LF assay devices have the advantage of being rapid, simple, largely automated, and cost effective. Relative ease of manufacture, long shelf life, and ease of use by the customer are some further advantages that make LF assay devices very attractive.
LF assay devices are often used as diagnostic devices where the main focus is on qualitative systems which provide an easy yes or no answer. Currently however, there is an increasing demand for more sensitive, quantitative and also multiplexing measurements which require the implementation of reader systems.
The most common sensing method used in LF assay devices is optical sensing, often by visual inspection, to detect a colorimetric, fluorescent or other visual change as a result of the assay. Reagent labels used to achieve such change include colloidal gold, latex particles, carbon, fluorescent molecules, and chemiluminescent molecules. Enzymatic reactions can be applied subsequently to amplify the signal. The main problems associated with visual detection of labels are: the relatively poor limits of detection in the absence of amplification, the inability to obtain kinetic information from the assay, and the lack of quantitative information gained from the assay. There is a distinct need for alternatives to visual characterization that can provide quantitative real time analysis while maintaining the simple, rapid, cost effective device that makes LF tests so appealing.
Ideally LF tests are run directly with a patient sample, without the need for purification, as this reduces the time, complexity and cost associated with processing prior to analysis. Unfortunately, the large variety of biomolecules in a raw sample increases the chance of nonspecific cross reactivity within the assay, which can cause a false positive reading as illustrated in FIG. 1. While this can be prevented through development of better, more specific, probe molecules, the development process thereof is expensive and laborious and is not guaranteed to eliminate cross-reactivity completely. However, it is well known that reaction kinetics and binding strengths differ between specific and non-specific binding interactions. Furthermore, these binding characteristics are susceptible to change through local environmental factors including temperature, pH, and ionic strength. By controllably varying local environmental parameters, it is possible to better characterize a detected interaction as either specific or non-specific.
Some other key factors that affect the signal produced in a LF assay device include temperature and ionic strength (including pH) of the solution or local environment. Including sensors and actuators that measure and influence such conditions allows for controllably varying the local environmental parameters in order to provide a more accurate qualitative and/or quantitative diagnostic device.
A biosensor providing a multiple site testing platform was described in US Published Patent Application US 2011/0091870, wherein the multiple sites in the biosensor could be subjected to different reaction conditions to modulate the binding of the biomolecular analyte (for example proteins) to the probe molecule. For example the signal detected in a biosensor having four sites also can have several components e.g four. These four terms correspond to for example the concentration of the biomarker of interest, the concentration of analytes in the sample that bind non-specifically to primary antibody sites and prevent the biomarker to bind, the concentration of the analytes in the sample that form a sandwich and produce a false signal, and finally the concentration of the analytes in the sample that physisorb to the surface and produce a false signal. Each term is also proportional to a binding efficiency factor, αij, which is a function of the molecular affinity and other assay conditions, e.g., flow speed. By controlling the condition in each site separately, different sites will exhibit different efficiency factors. Accurate measurement of the signal at each site will result in multiple equations and multiple unknowns—for example:
  {                                                        S              1                        =                                                            α                  11                                ⁢                                  C                  ag                                            -                                                α                  12                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    1                                                              +                                                α                  13                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    2                                                              +                                                α                  14                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    3                                                                                                                                      S              2                        =                                                            α                  21                                ⁢                                  C                  ag                                            -                                                α                  22                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    1                                                              +                                                α                  23                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    2                                                              +                                                α                  24                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    3                                                                                                                                      S              3                        =                                                            α                  31                                ⁢                                  C                  ag                                            -                                                α                  32                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    1                                                              +                                                α                  33                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    2                                                              +                                                α                  34                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    3                                                                                                                                      S              4                        =                                                            α                  41                                ⁢                                  C                  ag                                            -                                                α                  42                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    1                                                              +                                                α                  43                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    2                                                              +                                                α                  44                                ⁢                                  C                                      j                    ⁢                                                                                  ⁢                    3                                                                                            ⇒          C      ag      where Cag corresponds to the targeted biomolecular analyte concentration and Cj1, Cj2, Cj3 correspond to the total concentration of molecules which result in different terms in background signal, as shown in FIG. 2.
Accurate control of assay conditions at different sites, which allows large changes in the binding efficiency factors, results in an array of data from which a quantitative or more accurate qualitative determination of the biomolecular analyte of interests can be obtained. In addition, using electrical and electrochemical detection on a lateral flow assay device could significantly improve current point of care diagnostics.